The development of hemoglobin based blood substitutes continues to command commercial attention, and recent developments have shown that hemoglobin from mammalian blood cells, after suitable modification such as intramolecular crosslinking and in some instances polymerization, shows great promise as the basis of a blood substitute. As development has proceeded, however, the requirements for purity of the hemoglobin have steadily increased. At one time it was believed that hemoglobin simply needed to be stroma free, a condition achieved by washing and gentle lysing of the red blood cells, followed by filtration of the lysate. Subsequently, it was found that the presence of trace residues of impurities such as phospholipids led to more specific reactions, e.g. vasoconstriction, to the product in animal trials. Even after the product has been subjected to several diafiltration steps, it still contains unacceptably high traces of potentially harmful impurities such as erythrocyte enzymes, modified and variant forms of hemoglobin, phospholipids and surface antigens.
A hemoglobin-based blood substitute needs to be based either on a single hemoglobin species, or, if more than one species is present, a carefully controlled composition of known hemoglobin species. Accordingly, a successful hemoglobin purification process needs to be capable of separating one hemoglobin form from another, as well as separating the desired hemoglobin form from contaminating red blood cell such components such as erythrocyte enzymes, proteins, phospholipids and antigens.
Chemical crosslinking of hemoglobin for the preparation of the basis of a blood substitute commonly produces a mixture of hemoglobin species. These should subsequently be separated. Since they are in many cases of almost identical molecular weight and chemical composition, their separation presents difficulties.
Art example of a crosslinking reagent which produces a mixture of hemoglobin species, some crosslinked between certain pairs of positions on globin chains and others between other such positions, along with modified, uncrosslinked material and unchanged starting material, is the trig acyl (methyl phosphate) ester of 1,3,5-benzenetricarboxylic acid (TMMP), as disclosed in U.S. Pat. No. 5,250,665 Kluger et. al., issued Oct. 5, 1993.
Chromatographic methods have been applied to the purification of hemoglobin solutions. U.S. Pat. No. 4,925,474 Hsia et. al. describes the application of the techniques of affinity chromatography to hemoglobin purification, using columns in which a ligand showing preferential chemical binding affinity to the DPG site of hemoglobin was bound to the stationary phase of the column.
Ion exchange chromatographic techniques have also been applied to hemoglobin purification. The basic principles of the techniques of ion exchange chromatography are well known. A mixture of different species in a solution is applied to a suitably prepared ion exchange column. Each of the species in the mixture has a different affinity for the chemical reactant groups on the column. By varying the conditions on the column, e.g. the pH of the solution, the individual species can be arranged to bind or to elute from the column selectively, so as to separate one species individually from the mixture. The application of the technique to the purification of proteins such as hemoglobin is economically unattractive, except when used for small scale operations and analytical work. When hemoglobin is to be purified on a manufacturing scale, for use for example as an oxygen carrying resuscitative fluid (blood substitute), the technique, as conventionally applied, is impractical. The amounts of hemoglobin to be absorbed on and subsequently eluted from a chromatography column are so large that the column size requirements become impractically large and expensive.
Christensen et al., J. Biochem. Phys. 17 (1988), 143-154, reported the chromatographic purification of human hemoglobin. The methodology used represented a standard ion exchange chromatographic approach that did not provide opportunities for economical scale-up to production levels.
Winslow and Chapman, "Pilot-Scale Preparation of Hemoglobin Solutions," Methods in Enzymology," Vol. 231 p3 (1994) describe the preparation of stroma free hemoglobin (SFH), highly purified hemoglobin A0 and crosslinked hemoglobin using outdated human blood as starting material. The SFH is prepared by filtration, The Hemoglobin A0 is prepared from SFH by process scale chromatography using a strong anion-exchange medium and an ionic strength gradient.
U.S. Pat. No. 5,084,588 Rausch and Feola (Biopure), describes standard anion and cation exchange chromatography methods for application to separation and purification of hemoglobin. In the case of anion exchange chromatography, three standard approaches are listed in this patent:
a) binding of the Hb at elevated pH, and elution with a descending pH gradient or step gradient of lower pH; PA1 b) binding of the Hb at high pH, low ionic strength and elution with a salt gradient; PA1 c) loading under pH conditions where the hemoglobin does not bind to the anion exchanger, but passes through the column unretained, while the impurities (more acidic contaminants) are captured on the column.
Approaches a) and b) have been extensively documented, but are not attractive for large scale production, owing to the limitation of low loading capacities necessary to achieve sufficient resolution of the hemoglobin products. These loading capacities are routinely only 20-30 mg/ml, and dictate prohibitively large and expensive columns for commercial scale purification of hemoglobin. For example, a single 50 gm dose of final hemoglobin-based oxygen carrier (HBOC) would require a column of 1.5-2.5 liters.
Whilst approach c) would appear on the surface to be the most practicable, it turns out in practice that the chromatographic properties of normal human adult, unmodified hemoglobin Ao and some of the major contaminants, such as HbAlc, are not sufficiently distinct for practical application of this approach.
The standard approaches to cat ion exchange chromatography of mammalian hemoglobin have similar limitations.
U.S. Pat. No. 5,084,588 also discloses bovine hemoglobin solutions in which more than 99.9% of the protein present is bovine hemoglobins, but does not present identifying data for the protein constituents.